CN109939116B - Use of ROCK2 inhibitor for preparing medicine - Google Patents
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- CN109939116B CN109939116B CN201711383699.4A CN201711383699A CN109939116B CN 109939116 B CN109939116 B CN 109939116B CN 201711383699 A CN201711383699 A CN 201711383699A CN 109939116 B CN109939116 B CN 109939116B
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Abstract
The present invention provides the use of a ROCK2 inhibitor in the manufacture of a medicament for the treatment or prevention of glioma. The medicine can effectively prevent or treat drug-resistant glioma, in particular temozolomide-resistant glioma.
Description
Technical Field
The invention relates to the field of biomedicine, in particular to application of a ROCK2 inhibitor in preparation of a medicine.
Background
Temozolomide (TMZ), one of the only clinically available alkylating agents that can effectively penetrate the blood brain barrier, has long been used in tumor chemotherapy for gliomas since its discovery. Due to the wide application of the medicament, the glioma is easy to generate a medicament resistance phenomenon aiming at temozolomide clinically. The currently determined mechanisms for regulating and controlling the resistance of glioma to temozolomide mainly include methylation of O6-methylguanine DNA methyltransferase (O6-methylguanine DNA-transferase, MGMT), hyperfunction of genome repair function and the like, but no effective treatment scheme exists for the regulation and control modes, and further understanding of the mechanism of glioma resistance to temozolomide and exploring a new drug treatment scheme are needed.
Fasudil (Fasudil), also known as Fasudil, is an intracellular calcium antagonist, a specific selective RHO kinase inhibitor, marketed in china and japan. The composition is clinically used for treating, preventing and improving vasospasm caused by various reasons, selectively dilating spastic blood vessels and improving the ability of ischemia of heart and brain; meanwhile, the cerebral perfusion can be improved, and the anti-hypoxia capability of the brain can be enhanced; inhibit brain nerve cell damage, promote neuron axon growth; alleviating inflammatory response of affected brain cell tissue. Fasudil hydrochloride is mainly used for improving ischemic cerebrovascular disease symptoms caused by cerebral vasospasm and the like after subarachnoid hemorrhage.
Disclosure of Invention
The present application is based on the discovery and recognition by the inventors of the following facts and problems:
unless otherwise stated, fasudil is a compound represented by formula I, and temozolomide is a compound represented by formula II.
Glioma is easy to generate a drug resistance phenomenon aiming at temozolomide clinically, and an effective treatment scheme is not available at present. The inventor surprisingly finds that the clinically commonly used ROCK2 inhibitor is fasudil which is an intracellular calcium ion antagonist and can be used for treating glioma to a certain extent, and when the fasudil is combined with temozolomide, the fasudil can be used for treating drug-resistant glioma to a certain extent, and particularly, when the fasudil is combined with the temozolomide, the fasudil has a better treatment effect on the temozolomide-resistant glioma. Based on the findings of the above problems, the inventors have proposed for the first time the use of fasudil in the preparation of a medicament, and have proposed for the first time a pharmaceutical composition combining fasudil with temozolomide.
In a first aspect of the invention, the invention provides the use of a ROCK2 inhibitor in the manufacture of a medicament. According to an embodiment of the invention, the medicament is for the treatment or prevention of glioma. The inventor firstly discovers that the temozolomide can be sensitized by inhibiting the activity of ROCK2, and the proliferation of temozolomide resistant glioma can be inhibited in vitro and in vivo. The medicament provided by the embodiment of the invention can treat or prevent glioma to a certain extent, has a certain treatment or prevention effect on drug-resistant glioma, and particularly has a stronger treatment or prevention effect on temozolomide-resistant glioma.
According to an embodiment of the present invention, the above-mentioned use may further include at least one of the following additional technical features:
according to embodiments of the present invention, the ROCK2 inhibitor comprises a compound of formula I, or a stereoisomer, tautomer, enantiomer, nitrogen oxide, hydrate, solvate, metabolite, pharmaceutically acceptable salt or prodrug of a compound of formula I,
the inventor firstly uses fasudil which is commonly used in clinic for the treatment of glioma, and surprisingly finds that the fasudil can inhibit the growth of the glioma to a certain extent. The medicament according to the embodiment of the invention can treat or prevent glioma to some extent.
According to an embodiment of the invention, the glioma is a drug resistant glioma. The inventors have surprisingly found that fasudil is effective to some extent in the treatment or prevention of drug-resistant gliomas.
According to an embodiment of the invention, the drug-resistant glioma is a temozolomide-resistant glioma. The inventor finds that the therapeutic or preventive effect of fasudil on temozolomide-resistant glioma is better than that of other drug-resistant glioma.
According to an embodiment of the invention, the temozolomide resistant glioma is a temozolomide resistant glioma constructed from C6, U251, U87. It is noted that C6 is a rat glioma cell line, U251 is a human glioma cell line, and U87 is a human glioma cell line. The inventor finds that fasudil has better treatment or prevention effect on temozolomide-resistant glioma constructed by C6, U251 and U87.
According to an embodiment of the present invention, there is further included a compound of formula II or a stereoisomer, a tautomer, an enantiomer, a nitrogen oxide, a hydrate, a solvate, a metabolite, a pharmaceutically acceptable salt or a prodrug of the compound of formula II,
the invention provides a method for preventing or treating glioma by combining fasudil and temozolomide for the first time, and the inventor surprisingly discovers that the combination of fasudil and temozolomide has a certain effect on drug-resistant glioma, and when the glioma is temozolomide-resistant glioma, the effect is better, the treatment or prevention effect on the drug-resistant glioma constructed by C6, U251 and U87 is optimal, and the effect of inhibiting the drug-resistant glioma cells by using the fasudil alone or the temozolomide alone is not obvious by adopting the combination administration of fasudil and temozolomide to inhibit the growth of the drug-resistant glioma cells. According to the application of the embodiment of the invention, the combination of fasudil and temozolomide can obviously inhibit drug-resistant glioma and can effectively treat glioma, particularly drug-resistant glioma, especially temozolomide-resistant glioma.
According to an embodiment of the present invention, the mass ratio of the stereoisomer, tautomer, enantiomer, nitrogen oxide, hydrate, solvate, metabolite, pharmaceutically acceptable salt or prodrug of the compound represented by formula I to the stereoisomer, tautomer, enantiomer, nitrogen oxide, hydrate, solvate, metabolite, pharmaceutically acceptable salt or prodrug of the compound represented by formula II is 1: (1-12). The inventors found that, while decreasing the amount of fasudil is not effective in inhibiting the proliferation of temozolomide-resistant glioma without changing the amount of temozolomide drug, when the mass ratio of fasudil to temozolomide is 1: (1-12), the killing power to glioma cells is strongest.
In a second aspect of the invention, a pharmaceutical composition is provided. According to an embodiment of the present invention, the pharmaceutical composition is used for treating glioma, and comprises a ROCK2 inhibitor and other drugs for treating glioma. The inventor firstly discovers that the temozolomide can be sensitized by inhibiting the activity of ROCK2, and the proliferation of temozolomide resistant glioma can be inhibited in vitro and in vivo. The pharmaceutical composition provided by the embodiment of the invention can treat or prevent glioma to a certain extent, and has a certain treatment or prevention effect on drug-resistant glioma, and particularly has a stronger treatment or prevention effect on temozolomide-resistant glioma.
According to an embodiment of the present invention, the above pharmaceutical composition may further comprise at least one of the following additional technical features:
according to embodiments of the present invention, the ROCK2 inhibitor comprises a compound of formula I, or a stereoisomer, tautomer, enantiomer, nitrogen oxide, hydrate, solvate, metabolite, pharmaceutically acceptable salt or prodrug of a compound of formula I,
the inventor firstly proposes that fasudil is combined with other drugs for treating glioma to treat glioma.
According to an embodiment of the invention, the glioma is a drug resistant glioma. The inventor surprisingly found that the pharmaceutical composition also has a certain degree of inhibition on drug-resistant glioma.
According to an embodiment of the invention, the glioma is a temozolomide resistant glioma. The inventor finds that the treatment or prevention effect of the pharmaceutical composition on the temozolomide-resistant glioma is better than that of other drug-resistant gliomas.
According to an embodiment of the invention, the temozolomide resistant glioma is a temozolomide resistant glioma constructed from C6, U251, U87. The inventor finds that the pharmaceutical composition has the best treatment or prevention effect on temozolomide resistant glioma constructed by C6, U251 and U87.
According to the embodiment of the invention, the other medicines for treating glioma comprise lomustine, carmustine, nimustine, teniposide and temozolomide, and fasudil can be used together with the medicines for treating glioma, so that drug-resistant glioma can be inhibited to a certain extent.
According to an embodiment of the present invention, the other drug for treating glioma is temozolomide. The inventor firstly combines fasudil and temozolomide for preventing or treating glioma, and surprisingly discovers that the combination of fasudil and temozolomide has a certain effect on resistant glioma, the effect is better when the glioma is resistant to temozolomide, the effect on treating or preventing temozolomide-resistant glioma constructed by C6, U251 and U87 is optimal, and the effect on inhibiting drug-resistant glioma cells by singly using fasudil or singly using temozolomide is obvious without adopting the combination of fasudil and temozolomide for inhibiting the growth of drug-resistant glioma cells. According to the application of the embodiment of the invention, the combination of fasudil and temozolomide can obviously inhibit drug-resistant glioma and can effectively treat glioma, particularly drug-resistant glioma, especially temozolomide-resistant glioma.
According to an embodiment of the present invention, the mass ratio of the compound of formula I or the stereoisomer, tautomer, enantiomer, nitrogen oxide, hydrate, solvate, metabolite, pharmaceutically acceptable salt or prodrug of the compound of formula I to the temozolomide is 1: (1-12). The inventor finds that the reduction of the amount of fasudil can not effectively inhibit the proliferation of temozolomide-resistant glioma, and when the mass ratio of fasudil to temozolomide is 1: (1-12), the killing power to glioma cells is strongest.
According to an embodiment of the present invention, further comprising: a pharmaceutically acceptable excipient.
According to an embodiment of the present invention, the pharmaceutical composition is in the form of a tablet, injection, powder, elixir, capsule, suspension, syrup, pill or wafer.
According to an embodiment of the invention, the pharmaceutical composition is in the form of an injection. The fasudil hydrochloride is prepared into injection, the temozolomide is an oral medicine and is easy to decompose into a medicinal active ingredient in blood, and the medicinal composition according to the embodiment of the invention is prepared into injection.
Definitions and general terms
Reference will now be made in detail to certain embodiments of the invention, examples of which are illustrated by the accompanying structural and chemical formulas. The invention is intended to cover alternatives, modifications and equivalents, which may be included within the scope of the invention as defined by the appended claims. Those skilled in the art will recognize that many methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described herein. In the event that one or more of the incorporated documents, patents, and similar materials differ or contradict this application (including but not limited to defined terminology, application of terminology, described techniques, and the like), this application controls.
It will be further appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patents and publications referred to herein are incorporated by reference in their entirety.
The following definitions as used herein should be applied, unless otherwise indicated. For the purposes of the present invention, the chemical elements are in accordance with the CAS version of the periodic Table of the elements, and the handbook of chemistry and Physics, 75 th edition, 1994. In addition, general principles of Organic Chemistry can be referred to as described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausaltito: 1999, and "March's Advanced Organic Chemistry" by Michael B.Smith and Jerry March, John Wiley & Sons, New York:2007, the entire contents of which are incorporated herein by reference.
The articles "a," "an," and "the" as used herein are intended to include "at least one" or "one or more" unless otherwise indicated or clearly contradicted by context. Thus, as used herein, the articles refer to articles of one or more than one (i.e., at least one) object. For example, "a component" refers to one or more components, i.e., there may be more than one component contemplated to be employed or used in embodiments of the described embodiments.
The term "subject" as used herein refers to an animal. Typically the animal is a mammal. Subjects, e.g., also primates (e.g., humans, males or females), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, fish, birds, etc. In certain embodiments, the subject is a primate. In other embodiments, the subject is a human.
The term "patient" as used herein refers to humans (including adults and children) or other animals. In some embodiments, "patient" refers to a human.
The term "comprising" is open-ended, i.e. includes the elements indicated in the present invention, but does not exclude other elements.
"stereoisomers" refers to compounds having the same chemical structure but differing in the arrangement of atoms or groups in space. Stereoisomers include enantiomers, diastereomers, conformers (rotamers), geometric isomers (cis/trans), atropisomers, and the like.
"chiral" is a molecule having the property of not overlapping its mirror image; and "achiral" refers to a molecule that can overlap with its mirror image.
"enantiomer" refers to two isomers of a compound that are not overlapping but are in mirror image relationship to each other.
"diastereomer" refers to a stereoisomer having two or more chiral centers and whose molecules are not mirror images of each other. Diastereomers have different physical properties, such as melting points, boiling points, spectral properties, and reactivities. Mixtures of diastereomers may be separated by high resolution analytical procedures such as electrophoresis and chromatography, e.g., HPLC.
The stereochemical definitions and rules used in the present invention generally follow the general definitions of S.P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E.and Wilen, S., "Stereochemistry of Organic Compounds", John Wiley & Sons, Inc., New York, 1994.
Many organic compounds exist in an optically active form, i.e., they have the ability to rotate the plane of plane polarized light. In describing optically active compounds, the prefixes D and L or R and S are used to denote the absolute configuration of a molecule with respect to one or more of its chiral centers. The prefixes d and l or (+) and (-) are the symbols used to specify the rotation of plane polarized light by the compound, where (-) or l indicates that the compound is left-handed. Compounds prefixed with (+) or d are dextrorotatory. A particular stereoisomer is an enantiomer and a mixture of such isomers is referred to as an enantiomeric mixture. A50: 50 mixture of enantiomers is referred to as a racemic mixture or racemate, which may occur when there is no stereoselectivity or stereospecificity in the chemical reaction or process.
Any asymmetric atom (e.g., carbon, etc.) of a compound disclosed herein can exist in racemic or enantiomerically enriched forms, such as the (R) -, (S) -or (R, S) -configuration. In certain embodiments, each asymmetric atom has at least 50% enantiomeric excess, at least 60% enantiomeric excess, at least 70% enantiomeric excess, at least 80% enantiomeric excess, at least 90% enantiomeric excess, at least 95% enantiomeric excess, or at least 99% enantiomeric excess in the (R) -or (S) -configuration.
Depending on the choice of starting materials and methods, the compounds according to the invention may be present in the form of one of the possible isomers or of mixtures thereof, for example racemates and diastereoisomeric mixtures (depending on the number of asymmetric carbon atoms). Optically active (R) -or (S) -isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. If the compound contains a double bond, the substituents may be in the E or Z configuration; if the compound contains a disubstituted cycloalkyl group, the substituents of the cycloalkyl group may have cis or trans configurations.
Any resulting mixture of stereoisomers may be separated into pure or substantially pure geometric isomers, enantiomers, diastereomers, depending on differences in the physicochemical properties of the components, for example, by chromatography and/or fractional crystallization.
Racemization of any resulting final product or intermediate can be achieved by known methodsThe isomers are resolved into the optical enantiomers by methods familiar to the skilled worker, for example by separation of the diastereomeric salts obtained. The racemic product can also be separated by chiral chromatography, e.g., High Performance Liquid Chromatography (HPLC) using a chiral adsorbent. In particular, Enantiomers can be prepared by asymmetric synthesis, for example, see Jacques, et al, Enantiomers, racemes and solutions (Wiley Interscience, New York, 1981); principles of Asymmetric Synthesis (2) nd Ed. Robert E.Gawley,Jeffrey Aubé,Elsevier,Oxford,UK,2012)；Eliel,E.L.Stereochemistry of Carbon Compounds(McGraw-Hill,NY,1962)；Wilen,S.H.Tables of Resolving Agents and Optical Resolutions p.268(E.L.Eliel,Ed.,Univ.of Notre Dame Press,Notre Dame,IN 1972)； Chiral Separation Techniques:A Practical Approach(Subramanian,G.Ed.,Wiley-VCH Verlag GmbH&Co.KGaA,Weinheim,Germany,2007)。
The term "tautomer" or "tautomeric form" refers to structural isomers having different energies that can interconvert by a low energy barrier (low energy barrier). If tautomerism is possible (e.g., in solution), then the chemical equilibrium of the tautomer can be reached. For example, proton tautomers (also known as proton transfer tautomers) include interconversions by proton migration, such as keto-enol isomerization and imine-enamine isomerization. Valence tautomers (valenctautomers) include interconversion by recombination of some of the bonding electrons. A specific example of keto-enol tautomerism is the tautomerism of the pentan-2, 4-dione and 4-hydroxypent-3-en-2-one tautomers. Another example of tautomerism is phenol-ketone tautomerism. One specific example of phenol-ketone tautomerism is the tautomerism of pyridin-4-ol and pyridin-4 (1H) -one tautomers. Unless otherwise indicated, all tautomeric forms of the compounds of the invention are within the scope of the invention.
"metabolite" refers to the product of a particular compound or salt thereof that is metabolized in vivo. Metabolites of a compound can be identified by techniques well known in the art, and its activity can be characterized by assay methods as described herein. Such products may be obtained by administering the compound by oxidation, reduction, hydrolysis, amidation, deamidation, esterification, defatting, enzymatic cleavage, and the like. Accordingly, the present invention includes metabolites of compounds, including metabolites produced by contacting a compound of the present invention with a mammal for a sufficient period of time.
As used herein, "pharmaceutically acceptable salts" refer to organic and inorganic salts of the compounds of the present invention. Pharmaceutically acceptable salts are well known in the art, as are: berge et al, descriptive pharmacologically acceptable salts in detail in J. pharmaceutical Sciences,1977,66:1-19. Pharmaceutically acceptable non-toxic acid salts include, but are not limited to, salts of inorganic acids formed by reaction with amino groups such as hydrochlorides, hydrobromides, phosphates, sulfates, perchlorates, and salts of organic acids such as acetates, oxalates, maleates, tartrates, citrates, succinates, malonates, or those obtained by other methods described in the literature above, such as ion exchange. Other pharmaceutically acceptable salts include adipates, alginates, ascorbates, aspartates, benzenesulfonates, benzoates, bisulfates, borates, butyrates, camphorates, camphorsulfonates, cyclopentylpropionates, digluconates, dodecylsulfates, ethanesulfonates, formates, fumarates, glucoheptonates, glycerophosphates, gluconates, hemisulfates, heptanoates, hexanoates, hydroiodides, 2-hydroxy-ethanesulfonates, lactobionates, lactates, laurates, malates, malonates, methanesulfonates, 2-naphthalenesulfonates, nicotinates, nitrates, oleates, palmitates, pamoates, pectinates, persulfates, 3-phenylpropionates, picrates, pivalates, propionates, stearates, thiocyanate, p-toluenesulfonate, undecanoate, valerate, and the like. Salts obtained with appropriate bases include alkali metals, alkaline earth metals, ammonium and N + (C 1-4 Alkyl radical) 4 A salt. The present invention also contemplates quaternary ammonium salts formed from compounds containing groups of N. Water-soluble or oil-soluble or dispersion products can be obtained by quaternization. Alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Pharmaceutically acceptable salts further include suitable, non-toxic ammonium, quaternary ammonium salts and amine cations resistant to formation of counterions, such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, C 1-8 Sulfonates and aromatic sulfonates.
"solvate" of the present invention refers to an association of one or more solvent molecules with a compound of the present invention. Solvents that form solvates include, but are not limited to, water, isopropanol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate, acetic acid, and aminoethanol. The term "hydrate" refers to an association of solvent molecules that is water.
The term "treating" or "treatment" as used herein refers, in some embodiments, to ameliorating a disease or disorder (i.e., slowing or arresting or reducing the development of the disease or at least one clinical symptom thereof). In other embodiments, "treating" or "treatment" refers to mitigating or improving at least one physical parameter, including physical parameters that may not be perceived by the patient. In other embodiments, "treating" or "treatment" refers to modulating the disease or disorder, either physically (e.g., stabilizing a perceptible symptom) or physiologically (e.g., stabilizing a parameter of the body), or both. In other embodiments, "treating" or "treatment" refers to preventing or delaying the onset, occurrence, or worsening of a disease or disorder.
Pharmaceutically acceptable acid addition salts may be formed with inorganic and organic acids, for example, acetate, aspartate, benzoate, benzenesulfonate, bromide/hydrobromide, bicarbonate/carbonate, bisulfate/sulfate, camphorsulfonate, chloride/hydrochloride, chlorotheophylline, citrate, edisylate, fumarate, glucoheptonate, gluconate, glucuronate, hippurate, hydroiodide, isethionate, lactate, lactobionate, lauryl sulfate, malate, maleate, malonate, mandelate, methanesulfonate, methylsulfate, naphthoate, naphthalenesulfonate, nicotinate, nitrate, octadecanoate, oleate, oxalate, palmitate, pamoate, phosphate/biphosphate/dihydrogen phosphate, dihydrogenphosphate, and the like, Polysilonolactates, propionates, stearates, succinates, sulfosalicylates, tartrates, tosylates and trifluoroacetates.
Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like.
Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, sulfosalicylic acid, and the like.
Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases.
Inorganic bases from which salts can be derived include, for example, ammonium salts and metals of groups I to XII of the periodic table. In certain embodiments, the salts are derived from sodium, potassium, ammonium, calcium, magnesium, iron, silver, zinc, and copper; particularly suitable salts include ammonium, potassium, sodium, calcium and magnesium salts.
Organic bases from which salts can be derived include primary, secondary and tertiary amines, and substituted amines include naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like. Some organic amines include, for example, isopropylamine, benzathine (benzathine), choline salts (cholinate), diethanolamine, diethylamine, lysine, meglumine (meglumine), piperazine, and tromethamine.
The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound, basic or acidic moiety, by conventional chemical methods. In general, such salts can be prepared by reacting the free acid forms of these compounds with a stoichiometric amount of the appropriate base (e.g., Na, Ca, Mg, or K hydroxide, carbonate, bicarbonate, etc.), or by reacting the free base forms of these compounds with a stoichiometric amount of the appropriate acid. Such reactions are generally carried out in water or an organic solvent or a mixture of the two. Generally, where appropriate, it is desirable to use a non-aqueous medium such as diethyl ether, ethyl acetate, ethanol, isopropanol or acetonitrile. In, for example, "Remington's Pharmaceutical Sciences", 20 th edition, Mack Publishing Company, Easton, Pa., (1985); and "handbook of pharmaceutical salts: properties, Selection and application (Handbook of Pharmaceutical Salts: Properties, Selection, and Use) ", Stahl and Wermuth (Wiley-VCH, Weinheim, Germany, 2002) may find some additional lists of suitable Salts.
In addition, the compounds disclosed herein, including their salts, may also be obtained in the form of their hydrates or in the form of solvents containing them (e.g., ethanol, DMSO, etc.), for their crystallization. The compounds disclosed herein may form solvates with pharmaceutically acceptable solvents (including water), either inherently or by design; thus, the present invention is intended to include both solvated and unsolvated forms.
In another aspect, the present invention provides a pharmaceutical composition comprising a compound of the present invention, a pharmaceutically acceptable carrier, excipient, diluent, adjuvant, vehicle, or combination thereof. In some embodiments, the pharmaceutical composition may be in a liquid, solid, semi-solid, gel, or spray dosage form.
"combination" means a fixed combination or a kit of parts for combined administration in the form of a single dosage unit, wherein a compound disclosed herein and a combination partner may be administered separately at the same time or may be administered separately within certain time intervals, in particular such that the combination partners show a cooperative, e.g. synergistic, effect. The terms "co-administration" or the like as used herein are intended to encompass the administration of the selected combination partners to a single individual in need thereof (e.g. a patient), and are intended to encompass treatment regimens in which the substances are not necessarily administered by the same route of administration or simultaneously. The term "pharmaceutical combination product" as used herein denotes a product obtained by mixing or combining more than one active ingredient and includes both fixed and non-fixed combinations of active ingredients. The term "fixed combination" means that the active ingredients, such as the compounds disclosed herein and the combination partners, are administered to the patient simultaneously in the form of a single entity or dosage. The term "non-fixed combination" means that the active ingredients, such as the compounds of the disclosed compounds and the combination partners, are both administered to a patient as separate entities simultaneously, jointly or sequentially with no specific time limits, wherein the administration provides therapeutically effective levels of both compounds in the patient. The latter is also applicable to cocktail therapy, e.g. administering 3 or more active ingredients.
As used herein, the term "administering to a patient a compound as described above or a pharmaceutical composition as described above" means introducing a predetermined amount of a substance into the patient by some suitable means. The compound of formula (I) or the pharmaceutical composition of the present invention can be administered by any common route as long as it can reach the desired tissue. Various modes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, cortical, oral, topical, nasal, pulmonary and rectal, but the invention is not limited to these exemplified modes of administration. However, because of oral administration, the active ingredients of orally administered compositions should be coated or formulated to prevent degradation in the stomach. Preferably, the compound of formula (I) or the pharmaceutical composition of the present invention can be administered in an injectable formulation. Furthermore, the compounds of formula (I) or the pharmaceutical compositions of the present invention may be administered using a specific device for delivering the active ingredient to the target cells.
The administration frequency and dose of the pharmaceutical composition of the present invention can be determined by a variety of relevant factors including the type of disease to be treated, the administration route, the age, sex, body weight and severity of the disease of the patient and the type of drug as an active ingredient. According to some embodiments of the invention, the daily dose may be divided into 1, 2 or more doses in a suitable form, to be administered 1, 2 or more times over the entire period, as long as a therapeutically effective amount is achieved.
The term "therapeutically effective amount" refers to an amount of a compound sufficient to significantly ameliorate some of the symptoms associated with a disease or condition, i.e., to provide a therapeutic effect for a given condition and administration regimen. For example, in the treatment of gliomas, drugs or compounds that reduce, prevent, delay, inhibit, or arrest any symptom of the disease or disorder should be therapeutically effective. A therapeutically effective amount of a drug or compound need not cure a disease or condition, but will provide treatment for a disease or condition such that the onset of the disease or condition in an individual is delayed, prevented or prevented, or the symptoms of the disease or condition are alleviated, or the duration of the disease or condition is altered, or the disease or condition becomes less severe, or recovery is accelerated, for example.
The term "treatment" is used to refer to obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of complete or partial prevention of the disease or symptoms thereof, and/or may be therapeutic in terms of a partial or complete cure for the disease and/or adverse effects caused by the disease. As used herein, "treatment" encompasses treatment of a disease (primarily glioma, particularly a drug-resistant glioma) in a mammal, particularly a human, including: (a) preventing the onset of a disease (e.g., preventing glioma) or condition in an individual who is susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the development of diseases, such as drug-resistant gliomas; or (c) alleviating the disease, e.g., alleviating symptoms associated with the disease. As used herein, "treatment" encompasses any administration of a drug or compound to an individual to treat, cure, alleviate, ameliorate, reduce or inhibit a disease in the individual, including but not limited to the administration of a compound or pharmaceutical composition comprising formula (I) as described herein to an individual in need thereof.
Drawings
FIG. 1 is a graph showing inhibition of cell proliferation of temozolomide-resistant glioma cells by fasudil according to an embodiment of the present invention;
fig. 2 is a graph showing the proliferation inhibitory effect of different concentrations of fasudil in combination with temozolomide on temozolomide-resistant glioma cells C6R, wherein C6R represents the inhibition rate of temozolomide alone, according to an embodiment of the present invention;
fig. 3 is a graph of the inhibition of proliferation of temozolomide-resistant glioma cells U251R by different concentrations of fasudil in combination with temozolomide administration according to an embodiment of the present invention, wherein U251R represents the inhibition rate of temozolomide administered alone;
fig. 4 is a graph of the inhibition of proliferation of temozolomide-resistant glioma cells U87R by different concentrations of fasudil in combination with temozolomide administration according to an embodiment of the present invention, wherein U87R represents the inhibition rate of temozolomide administered alone;
fig. 5 is a graph showing that fasudil administered in combination with temozolomide according to an embodiment of the present invention inhibits the change of U251R axillary transplantation tumor volume, wherein U251R shows the change trend of tumor volume of a blank control group;
FIG. 6 is a graph of the effect of fasudil in combination with temozolomide on the weight of U251R axillary transplantable tumors, in accordance with an embodiment of the present invention;
FIG. 7 is a graph showing the effect of fasudil in combination with temozolomide on body weight of mice bearing U251R tumor cells, according to an embodiment of the present invention;
FIG. 8 is a graph showing the effect of fasudil in combination with temozolomide on the weight of the major organs of a mouse bearing U251R tumor according to an embodiment of the present invention;
FIG. 9 is a graph showing the effect of fasudil in combination with temozolomide on the survival rate of intracranial C6R transplanted tumor rats, according to an embodiment of the present invention;
FIG. 10 is a graph showing the effect of fasudil in combination with temozolomide administration on the body weight of intracranial C6R transplanted tumor rats, according to an embodiment of the present invention;
FIG. 11 is a graph showing the effect of fasudil in combination with temozolomide on the footprint of intracranial C6R tumor lesions, in accordance with an embodiment of the present invention;
FIG. 12 is a quantification of the effect of fasudil in combination with temozolomide on the footprint of intracranial C6R tumor lesions, according to an embodiment of the invention;
FIG. 13 is a graph showing the apparent activation of ROCK2 in temozolomide-resistant glioma cells following knockdown of ROCK2 (i.e., inhibition of ROCK2 protein expression) using small interfering RNA (siRNA) in response to temozolomide sensitivity in temozolomide-resistant glioma cells in accordance with an embodiment of the present invention;
FIG. 14 is a graph showing the change in sensitivity of 3 cell lines to temozolomide that activated ROCK2 in U251, U87, C6 cells using agonist hemolytic phosphatidic acid (LPA) of ROCK2, according to an embodiment of the present invention; and
fig. 15 is a graph of the change in phosphorylation of ROCK2 and expression of ABCG2 after administration of different concentrations of fasudil in accordance with an embodiment of the invention.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.
Example 1 experiment of proliferation inhibition of fasudil in combination with temozolomide on human glioma-resistant temozolomide cells U251R and U87R, and rat glioma-resistant temozolomide cell C6R
1. Experimental Material
(1) Medicine
Temozolomide bulk drug powder (C) 6 H 6 N 6 O 2 ) From Jili chemical Co., Ltd, New zone, Changzhou, as white powder, protected from light. A20 mM stock solution was prepared using dimethyl sulfoxide (DMSO), and stored at-20 ℃. Before use, DMEM culture solution containing 10% fetal calf serum, 100U/mL penicillin and 100mg/L streptomycin is prepared into required concentration, and the final concentration of DMSO is controlled below 1 per thousand.
Fasudil hydrochloride powder (Fasudil (HA-1077) HCl, C 14 H 17 N 3 O 2 S.hcl), available from hailan chemicals ltd, supra-sea, as a white powder. The stock solution was prepared at 100mM using Phosphate Buffered Saline (PBS) and stored at-80 ℃. The required concentration was prepared just before use in DMEM medium containing 10% fetal calf serum, 100U/mL penicillin and 100mg/L streptomycin.
(2) Reagent
DMEM culture solution: a bag (13.5g) of DMEM medium powder (GIBCO, USA) was dissolved in 1000mL of sterilized triple distilled water, and NaHCO was used 3 Adjusting the pH value to 7.3-7.4, filtering and sterilizing by a cylindrical filter, and storing in a refrigerator at 4 ℃. Before use, 10% fetal calf serum, 100U/mL penicillin and 100mg/L streptomycin are added.
② fetal bovine serum: hangzhou Sijiqing bioengineering company product. Inactivating in 56 deg.C water bath for 30min, subpackaging, and storing in-20 deg.C low temperature refrigerator.
③ PBS buffer solution: weighing NaCl 8.0g,KCl 0.20g、Na 2 HPO 4 ·H 2 O 1.56g、KH 2 PO 4 2.0g, dissolved in 1000mL of triple distilled water, autoclaved, and stored in a refrigerator at 4 ℃.
0.25% pancreatin: weighing pancreatin 0.25g, dissolving in 100ml PBS buffer solution, filtering and sterilizing.
Fifth, DMSO: Sigma-Aldrich, USA (St. Louis, Mo).
Sixthly, MTT solution: 50mg of MTT powder (Bioshrp) was weighed out, prepared with 10ml PBS, and sterilized by filtration through a 0.22 μm filter.
(3) Cell line
The temozolomide-resistant cell lines C6R, U251R and U87R are constructed by using a rat glioma cell line C6, a human glioma cell line U251 and a human glioma cell line U87 provided by a biochemical and cell biology research institute of Shanghai institute of Life sciences of China academy of sciences. IC according to C6, U251, U87 50 Set up IC 50 1/16 is the initial drug-resistant dose induction, TMZ with the concentration is added at the initial point of logarithmic proliferation phase of the cells, the cells are observed after 72h, if the cells die in a large amount, a new culture medium is replaced, the concentration of the TMZ is kept unchanged, and the culture medium with the concentration of the TMZ is replaced every 72h until the cells are confluent and grow to 80% density; if no cell death occurs after TMZ is added for 72h for the first time, carrying out normal passage, adding a TMZ culture medium containing the initial induction drug-resistant dose concentration, culturing for 72h, and if cell death occurs, treating according to the above description, if no cell death occurs, adding a drug with 2 times of the initial induction drug-resistant dose concentration. After the cells treated in the steps grow to 80% of density, the cells are normally passaged, and TMZ with one-time dosage is increased after passage; repeating the above steps until the concentration of the medicine is increased to IC 50 Then, the TMZ was raised at a concentration of 10. mu.M for each cycle; when reaching IC 50 1.5 times higher, 5 μ M of TMZ was again raised per dosing cycle; final addition of TMZ concentration to IC 50 At 2 times higher, TMZ resistance is terminated. Cells were tested for TMZ-resistant properties. The entire TMZ-resistant process lasts approximately 6-10 months.
C6, U251 and U87 are cultured by using DMEM culture solution containing 10% fetal bovine serum; all TMZ-resistant cell lines were cultured in one-to-two manner, each resistant cell line being fineAdding the cells into the daily culture solution, and adding respective maternal cell line TMZ IC 50 The 1/2 concentration of (a) is taken as the drug concentration at which the TMZ resistance property is maintained.
2. Laboratory apparatus
(1) YJ-875 type medical purification workbench: suzhou purification plant.
(2)3111 type water-jacketed CO2 incubator: product of Thermo electron corporation, usa.
(3) Model OlympusIX51 inverted fluorescence microscope: a product of Olympus corporation of japan.
(4) An electronic balance: product of Beijing Sidolis Instrument systems, Inc.
(5) ZW-A type micro oscillator: tan, Tan.
(6) 35800 enzyme linked immunosorbent assay device, available from BioTeK corporation of usa.
3. Experiment of drug effect
Adopting tetramethyl azoazolium salt (MTT) experiment to investigate the inhibiting effect and IC of fasudil, temozolomide, fasudil and temozolomide on different temozolomide resistant glioma cells 50 The value is obtained.
C6R, U251R, U87R cells in logarithmic growth phase were digested with 0.25% pancreatin, centrifuged, resuspended and counted to make a cell suspension at 5X 10 4 Adding the cell concentration into a 96-well enzyme label plate with 100 mu L of each well, arranging 3 wells, placing at 37 ℃ and 5% CO 2 Culturing in an incubator for about 12h until the cells are completely attached to the wall. The dosing treatment was carried out as follows:
a first group: fasudil is added, and the final concentration in each hole is 1, 10, 50, 100 and 200 mu M;
second group: adding temozolomide to the solution at the final concentration of 125, 250, 500, 1000 or 2000 mu M in each well;
third group: the temozolomide and low-concentration fasudil are added for combined administration, the final concentration of the temozolomide in each hole is 125, 250, 500, 1000 and 2000 mu M, and the final concentration of the fasudil is 0.4 mu M;
and a fourth group: the temozolomide and fasudil with medium concentration are added for combined administration, the final concentration of the temozolomide in each hole is 125, 250, 500, 1000 and 2000 mu M, and the final concentration of the fasudil is 2 mu M;
and a fifth group: the temozolomide and high-concentration fasudil are added for combined administration, the final temozolomide concentration in each hole is 125, 250, 500, 1000 and 2000 mu M, and the final fasudil concentration is 10 mu M.
Control group: cells were added only to the same blank medium as the dosed group.
Adding medicine into each hole, incubating for 48h, adding 20 mu L of 5mg/mL MTT solution, incubating for 4h at 37 ℃ in the dark, discarding all supernatants, adding 100 mu L DMSO into each hole, oscillating for 2-3 min on a micro oscillator, and determining the absorbance value A at the wavelength of 570nm by using an enzyme linked immunosorbent assay detector after crystals are completely dissolved. The larger the value of A, the larger the number of viable cells. The experiment is repeated 3 times, and the growth inhibition rate of the drug on the cells is calculated according to the value A: growth inhibition rate of 1-A Medicine adding set /A Control group 。
Calculating half inhibitory concentration IC according to Sunshima's synthesis method (modified Kouzhou's method) 50 (i.e., the concentration of drug required to inhibit 50% of cell growth).
The experimental results are shown in figures 1-4 and table 1, compared with the single administration of fasudil or temozolomide, the combination administration of fasudil and temozolomide can obviously inhibit the proliferation of drug-resistant cells (p is less than 0.05), and the fasudil can obviously increase the IC of temozolomide-resistant cells on temozolomide 50 . The fasudil is proved to achieve the effect of killing drug-resistant cells by increasing the sensitivity of the drug-resistant cells to temozolomide.
Table 1: IC of temozolomide after administration of different concentrations of fasudil U251R, U87R, C6R 50
This is marked by p <0.05 compared to untreated group * P <0.01 marker ** 。
Example 2 investigation of the therapeutic Effect of fasudil in combination with temozolomide on glioma-resistant temozolomide-transplanted tumors in nude mice
1. Experimental Material
(1) Medicine
Temozolomide was dissolved in PBS buffer at pH 4.0 and administered by intraperitoneal injection at an interval of 25mg/kg per nude mouse.
Fasudil was dissolved in PBS buffer at pH 4.0, and administered by intraperitoneal injection at an interval of 20mg/kg per nude mouse.
The administration volumes of temozolomide and fasudil are both 0.01 mL/g.
(2) Reagent
DMEM culture solution: a bag (13.5g) of DMEM medium powder (GIBCO, USA) was dissolved in 1000mL of sterilized triple distilled water, and NaHCO was used 3 Adjusting the pH value to 7.3-7.4, filtering and sterilizing by a cylindrical filter, and storing in a refrigerator at 4 ℃. Before use, 10% fetal calf serum, 100U/mL penicillin and 100mg/L streptomycin are added.
② fetal bovine serum: hangzhou Chinese ilex bioengineering company product. Inactivating in 56 deg.C water bath for 30min, subpackaging, and storing in-20 deg.C low temperature refrigerator.
(iii) PBS buffer: weighing 8.0g of NaCl, 0.20g of KCl and Na 2 HPO 4 ·H 2 O 1.56g、KH 2 PO 4 2.0g, dissolved in 1000mL of triple distilled water, autoclaved, stored in a refrigerator at 4 ℃, and the pH is adjusted to 4.0 by hydrochloric acid when preparing the medicine.
0.25% pancreatin: pancreatin 0.25g was weighed, dissolved in 100mL of PBS buffer, and sterilized by filtration.
(3) Cell line
Temozolomide-resistant cell line U251R (constructed as in example 1) was cultured in DMEM medium containing 10% fetal bovine serum.
(4) Laboratory animal
Source, strain: BALB/c-nu nude mice, provided by the model animal institute of Nanjing university (license number: SCXK (Su) 2010-0001). The age in days: 35-40 days, body weight: 18-24g, sex: half a female and half a male.
2. Experimental methods
Taking a temozolomide resistant glioma cell strain U251R in logarithmic growth phase, and preparing the temozolomide resistant glioma cell strain into 2 x 10 cells under aseptic condition 7 0.2mL of the cell suspension was inoculated to the nudeMice right axilla were subcutaneous. Measuring the long diameter and the short diameter of the transplanted tumor by using a vernier caliper for the transplanted tumor of the nude mouse until the tumor grows to 100mm 3 Animals were then randomized into groups, each treatment group was as follows: blank control group: 10 (solvent control, i.p., PBS buffer at pH 4.0 equal volume per day); 10 temozolomide groups (temozolomide, 25mg/kg, i.p., temozolomide per day, solvent per day); 10 fasudil groups (fasudil, 20mg/kg, i.p., fasudil per day, solvent per day); temozolomide and fasudil administration groups (temozolomide, 25mg/kg, fasudil, 20mg/kg, i.p., one day fasudil, one day temozolomide) 10. The volume administered was 0.01 mL/g. The tumor diameter is measured 1 time every 2-3 days, and the anti-tumor effect of the test substance is dynamically observed. After 21 days, the mice were sacrificed and the tumor mass was surgically removed and weighed.
The Tumor Volume (TV) is calculated as: TV 1/2 × a × b 2 Wherein a and b represent a long diameter and a wide diameter, respectively.
3. Results
The treatment results of fasudil and temozolomide administration on human glioma temozolomide-resistant U251R nude mouse transplanted tumor are shown in fig. 5-8. As shown in fig. 5, compared with the blank control group, temozolomide group and fasudil group, the tumor volume of the temozolomide and fasudil group was significantly different from that of the other groups at 15 days, the tumor volume was significantly smaller than that of the other groups, and the tumor volume at each time point thereafter was significantly smaller than that of the other treatment groups, and there was a statistical difference (p <0.05, p < 0.01; p < 0.05; p < 0.01; fasudil group, & p < 0.01). There were no statistical differences between the remaining groups. As shown in FIG. 6, the tumor weight of the combination-administered group was significantly less than that of the other groups and was statistically different (p <0.05 and p <0.01 in comparison with the blank control group, # p <0.05 and p <0.01 in comparison with the temozolomide group, # p <0.05 and p <0.01 in comparison with the fasudil group, & p <0.05 and p <0.01 in comparison with the fasudil group). As can be seen from FIG. 7, there was no significant difference between the body weights of the animals in each group; as shown in fig. 8, there was no significant difference between the groups in the weight of the major animal organs, indicating that the toxicity of the combination was low. In conclusion, fasudil and temozolomide have obvious growth inhibition effect on xenograft tumor of human glioma temozolomide-resistant cell U251R nude mouse.
Example 3 investigation of the therapeutic Effect of fasudil in combination with temozolomide on glioma-resistant temozolomide rat orthotopic transplantation tumor
1. Experimental Material
(1) Medicine
Temozolomide, dissolved in PBS pH 4.0, was administered intraperitoneally at 35mg/kg every other day per rat.
Fasudil, dissolved in PBS, was administered intraperitoneally at 30mg/kg every other day per rat.
(2) Reagent
DMEM culture solution: a bag (13.5g) of DMEM medium powder (GIBCO, USA) was dissolved in 1000mL of sterilized distilled water, and NaHCO was used 3 Adjusting the pH value to 7.3-7.4, filtering and sterilizing by a cylindrical filter, and storing in a refrigerator at 4 ℃. Before use, 10% fetal calf serum, 100U/mL penicillin and 100mg/L streptomycin are added.
② fetal bovine serum: hangzhou Sijiqing bioengineering company product. Inactivating in 56 deg.C water bath for 30min, packaging, and storing in-20 deg.C low temperature refrigerator.
③ PBS buffer solution: weighing 8.0g NaCl, 0.20g KCl and Na 2 HPO 4 ·H 2 O 1.56g、KH 2 PO 4 2.0g, dissolved in 1000mL of triple distilled water, autoclaved, and stored in a refrigerator at 4 ℃.
0.25% pancreatin: weighing pancreatin 0.25g, dissolving in 100ml PBS buffer solution, filtering and sterilizing.
4% neutral formaldehyde: weighing 4g of paraformaldehyde powder, dissolving in 60mL of ultrapure water, adding NaOH into a solvent in a water bath kettle at 50 ℃ for assisting melting until the paraformaldehyde is completely dissolved, adjusting the pH value to 7.4 by using 36-38% of HCl, and fixing the volume to 100mL by using a volumetric flask.
(3) Cell line
Temozolomide-resistant cell line C6R (constructed as in example 1) was cultured in DMEM medium containing 10% fetal bovine serum.
(4) Laboratory animal
Source, strain: sprague Dawley rats, provided by the university of Nanjing institute of model animals (license number: SCXK (Su) 2010-0001). The age in days: 35-40 days, body weight: 190 + 210g, sex: the male and female are half.
2. Experimental method
(1) Grouping
Blank control group: 12, only one of the raw materials is used;
temozolomide group (temozolomide, 35mg/kg, i.p., temozolomide daily, solvent daily): 12, the number of the main components is 12;
fasudil group (fasudil, 25mg/kg, i.p., fasudil, one day solvent): 12, only one of the raw materials is used;
temozolomide combined fasudil administration group (temozolomide, 35mg/kg, fasudil, 25mg/kg, i.p., fasudil a day, temozolomide a day): and 12 are used.
(2) Molding method
Preparation of cells: after centrifugation, the cells were counted using PBS buffer pre-cooled to 4 ℃ containing 10 cells per 10. mu.L 6 Individual C6R cells were suspended at concentration and stored on ice.
② SD rat is anesthetized by 3% sodium isopentbarbital (0.4-0.6 mL/200g), fixed on an operating table, hair in the operating area is removed, the operating area is disinfected by iodophor, after the animal is determined to enter deep anesthesia state, an opening of about 1.0cm is opened along the midline of cranium by using an operating knife, meninges is scraped off gently, and cranium is drilled by a cranium with the diameter of 1.0mm at the position 1.0mm behind the bregma and 3.0mm at the right side of the midline. After punching, insert the sample injector gently into the hole, enter 6mm, exit 1mm, and gently push 10 μ L (10) 6 C6R cells) for 6min, stopping the needle for 2min after the sample injection is finished, slightly pulling out the sample injection needle, sealing the skull drill hole with bone wax, suturing the wound, applying a thermal insulation measure after the operation, and continuously applying 1.6 ten thousand units of penicillin sodium intramuscular injection to the rat for 3 days. Rats were observed 3 days post-operatively and after definitive no more animal death, animals were grouped into groups of 12 animals (hermaphrodite halves). Dosing began the second day after the group.
(3) Data processing
After 25 days of treatment, each group of SD rats is sacrificed, brain tissues are stripped, accessory structures are removed, and the SD rats are placed in 4% neutral formaldehyde for fixation. And (4) slicing the fixed sample, and carrying out HE staining to observe the tumor occupying lesion.
Measuring the tumor area in the section by using imageJ software, and calculating the relative tumor area.
Relative tumor area is treatment/blank.
3. Results
The results of fasudil combined with temozolomide treatment of intracranial C6R transplantable tumors are shown in fig. 9-12. As shown in fig. 9, the blank group showed animal death at day 7, while the temozolomide group showed animal death at day 5, and the fasudil group showed animal death after 9 days of continuous administration, and compared with the three groups, the temozolomide and fasudil combination group showed significantly longer time for death of the first animal, which is day 17 after administration; by the end of the treatment period (i.e., the survival rate of any group in this experiment was less than 50%), the survival rate of the combination group was significantly higher than that of the placebo, temozolomide, and fasudil groups, and there were statistical differences (p <0.05 in comparison with the placebo group, # p <0.05 in comparison with the temozolomide group, and # p <0.05 in comparison with the fasudil group). As shown in FIG. 10, animals in the combination group began to weigh significantly more than the other groups at day 17 and were statistically different from day 17 to day 25 (p <0.05 for the group with blank control and p <0.01 for the group with temozolomide and p <0.05 for the group with fasudil), indicating that the combination reduced the adverse effects of intracranial neoplastic lesions on animal physiology. As shown in fig. 11 and 12, the occupied area of the intracranial tumor in the combination group was significantly smaller than that of each of the other groups, and when the occupied area was counted, the area of the combination group was significantly smaller than that of each group, but was statistically different from that of the blank group and fasudil group, and was not statistically different from that of temozolomide group (p was less than 0.01 compared to the blank control group; p was less than 0.01 compared to temozolomide group, # was less than 0.01 compared to fasudil group, & was less than 0.01 compared to fasudil group). In conclusion, the fasudil and temozolomide combined administration has obvious growth inhibition effect on C6R intracranial transplantation tumors.
Example 4 inhibition of ROCK2 Activity sensitizes temozolomide in temozolomide-resistant cells
1. Experimental Material
(1) Medicament
When fasudil is used, PBS is used for dissolving the fasudil to prepare a mother solution with the concentration of 100 mM.
(2) Reagent
DMEM culture solution: a bag (13.5g) of DMEM medium powder (GIBCO, USA) was dissolved in 1000mL of sterilized distilled water, and NaHCO was used 3 Adjusting the pH value to 7.3-7.4, filtering and sterilizing by a cylindrical filter, and storing in a refrigerator at 4 ℃. Before use, 10% fetal calf serum, 100U/mL penicillin and 100mg/L streptomycin are added.
② fetal bovine serum: hangzhou Sijiqing bioengineering company product. Inactivating in 56 deg.C water bath for 30min, subpackaging, and storing in-20 deg.C low temperature refrigerator.
③ PBS buffer solution: weighing 8.0g of NaCl, 0.20g of KCl and Na 2 HPO 4 ·H 2 O 1.56g、KH 2 PO 4 2.0g, dissolved in 1000mL of triple distilled water, autoclaved, and stored in a refrigerator at 4 ℃.
0.25% pancreatin: weighing pancreatin 0.25g, dissolving in 100ml PBS buffer solution, filtering and sterilizing.
Protein antibody and other reagents: Anti-ROCK2, Abcam corporation, Specifications: 40 μ L, cat #: ab 125025; Anti-p-ROCK2(Tyr 722), Abcam, 100 μ L, cat #: ab 182648; high-sig ECL Western Blotting Substrate, Shanghai energy Biotechnology Ltd, Specification: 500mL, cargo number: 180-5001; biostep TM Prestained Protein Marker, shanghai sky biotechnology limited, specification: 200 μ L, cat No.: 180-6003; siRNA-control, Santa Cruz Biotechnology, Specification: 10 μ M, cat no: sc-37007; siRNA-ROCK2, humanized sequence: GCAgACAAgAAACgAAAUUUg, rat derived sequence: GUCUAUUAAUACUCGUCUA, synthesized by Shanghai; anti-ABCG2 from Bioworld, size 100. mu.L, cat #: BS 3482; PVDF membrane, Immobilon-P, Millipore, cat number: IPVH 00010.
(3) Cell line
Temozolomide-resistant cell lines U251R, U87R, and C6R (constructed as in example 1) were cultured in DMEM medium containing 10% fetal bovine serum.
2. Experimental method
(1) Total protein extraction
Extracting cell protein: cells treated as needed were washed 2 times with PBS. Scraping cells with a cell scraper, centrifuging for 10min at 500g, adding an appropriate amount of RIPA lysate containing 1% PMSF, suspending the cells, lysing for 1.5h on ice, centrifuging for 30min at 4 ℃, sucking supernatant, and determining protein content with Nano-100; then, proteins were normalized to the same concentration using RIPA lysate, 5 x SDS protein loading buffer was added, denatured at 100 ℃ for 8min, rapidly placed on ice for cooling, and centrifuged before use in a western blot.
(2) Western Blot experiment
SDS gels with different concentrations are configured according to the molecular weight of target protein, the electrophoresis conditions are that the concentration gel is at constant voltage of 75V, the separation gel is at constant voltage of 135V, and the electrophoresis time is about 2 hours.
Cutting a proper separation gel block according to the required protein molecular weight after electrophoresis is finished, and transferring the membrane by using a semi-dry or full-wet transfer membrane method.
Third, semi-dry transfer membrane method: cutting appropriate separation gel, spreading a sandwich structure from cathode to anode filter paper-gel-membrane-filter paper, carefully removing air bubbles, and filtering according to membrane area (cm) 2 ) Multiplying by a coefficient of 1.2(mA) to calculate the current, and carrying out constant current film transfer for 60-90 min. Full wet transfer membrane method: cutting out proper separation glue, paving a rotary membrane sandwich structure in the sequence of filter paper-glue-membrane-filter paper from the cathode to the anode, clamping a frame, placing the frame into a rotary membrane groove filled with a rotary membrane liquid, placing the rotary membrane groove into a barrel filled with an ice bag or a freezer at 4 ℃, and rotating the membrane for 2.5-4 hours at a constant current of 300 mA.
Fourthly, after the membrane transfer is finished, the PVDF membrane is sealed by a shaker for 2 hours at 4 ℃ and contains 3 percent calf serum albumin (BSA). Thereafter, the blocking solution was discarded, and the PVDF membrane was washed with PBST 3 times for 10min each. Primary antibody was added at the appropriate concentration and incubated overnight on a shaker at 4 ℃.
Fifthly, primary antibody is recovered every other day, PBST is used for cleaning, 10min each time, and 3 times are total. Adding the secondary antibody diluted by PBST, and incubating for 2h at a constant temperature of 4 ℃ in a shaking table. Discarding the secondary antibody after the incubation is finished, and washing the strip on a decolorizing shaker by using PBST for 10min each time for 3 times; and then dripping the ECL luminescent liquid which is prepared freshly onto the PVDF membrane to be detected, scanning the membrane in a gel imaging analysis system for imaging, and analyzing the result of the western blot.
(3) Data statistics and analysis
Cell experiments were performed in parallel, in triplicate, and all data were expressed as mean ± SD and statistically analyzed using SPSS19.0 software. Paired t test is used between the two groups; one-way ANOVA test was performed between groups. Small samples or non-normal distribution data were obtained using non-parametric Mann-Whitney U-test. P <0.05 indicates significant difference, and P <0.01 indicates very significant difference.
3. Results
Inhibition of ROCK2 can sensitize temozolomide-resistant cells to temozolomide sensitivity, with results shown in fig. 13-15. As shown in fig. 13, the inventors found that both the expression and activation (phosphorylation in the activated form) of ROCK2 were significantly increased in temozolomide-resistant glioma cells (a); whereas, after using small interfering rna (sirna) to knock down the expression of ROCK2 protein, temozolomide-resistant cells showed a significant increase in sensitivity to temozolomide (B and C, p <0.05, p <0.01 compared to temozolomide-resistant cell blanks). As shown in fig. 14, the inventors stimulated U251, U87, C6 cells with hemolytic phosphatidic acid (LPA), an agonist of ROCK2, and found that LPA significantly increased phosphorylation of ROCK2 (a), and that U251, U87, C6, under LPA stimulation, showed a significant decrease in sensitivity to temozolomide (B, p <0.05, p <0.01 compared to blank). As shown in fig. 15, phosphorylation of ROCK2 decreased significantly with increasing dose after administration of fasudil at different concentrations, and expression of ABCG2, which is one of important targets for tumor resistance, also decreased significantly. Taken together, inhibition of ROCK2 activity can sensitize temozolomide in temozolomide-resistant cells and down regulate ABCG2 expression.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are exemplary and not to be construed as limiting the present invention, and that changes, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (2)
1. Use of fasudil or a pharmaceutically acceptable salt thereof and temozolomide or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for the treatment or prevention of glioma;
the mass ratio of the fasudil or the pharmaceutically acceptable salt thereof to the temozolomide or the pharmaceutically acceptable salt thereof is 1: (1-12);
wherein the glioma is temozolomide resistant glioma.
2. The use of claim 1, wherein the temozolomide resistant glioma is a temozolomide resistant glioma constructed from C6, U251, U87.
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